Quantitation and identification of dimers in co-formulations

ABSTRACT

Methods and system for identification of dimer species using online chromatography and electrospray ionization mass spectrometry are provided. Also provided are methods and system for quantitation of heterodimer species using immunoprecipitation and liquid chromatography-mass spectrometry.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/750,845, filed on Jan. 23, 2020, which claims priority to and thebenefit of U.S. Provisional Patent Application No. 62/852,591, filed onMay 24, 2019, and also claims priority to and the benefit of ProvisionalPatent Application No. 62/796,794, filed on Jan. 25, 2019; the contentof which is incorporated herein by reference in its entirety.

FIELD

The invention generally pertains to a method and system foridentification of dimer species using online chromatography andelectrospray ionization mass spectrometry and quantitation ofheterodimer species using immunoprecipitation and liquidchromatography-mass spectrometry.

BACKGROUND

Protein biopharmaceutical products have emerged as important drugs forthe treatment of cancer, autoimmune disease, infection andcardiometabolic disorders, and they represent one of the fastest growingproduct segments of the pharmaceutical industry.

The strategy of co-formulating two or more therapeutic monoclonalantibodies (mAbs) and/or active proteins into one final drug product hasgained a lot of popularity recently offering several advantages,including increased efficacy, overall reduced adverse events andimproved patient convenience and compliance. Formulating two differentmAbs and/or active proteins in a single formulation may be problematicand involves choosing excipients and conditions that may represent acompromise. In addition to challenges for formulation development,co-formulated drugs also present significant challenges for analyticalcharacterization. For example, differentiation and quantitation ofdifferent dimer forms present in a co-formulated drug under normalstorage or stressed conditions may be challenging and often cannot beachieved by traditional methods such as size exclusion chromatography.

Protein biopharmaceutical products including co-formulated preparationsmust meet very high standards of purity. Thus, it may be important tomonitor any impurities in the co-formulated drug at different stages ofdrug development, production, storage and handling. Analytical methodsfor assays for characterization should display sufficient accuracy andresolution to detect and quantify the desired product. Direct analysiscan require isolation of the product in a sufficiently large amount forthe assay, which is undesirable and has only been possible in selectedcases.

There is a long felt need in the art for a method and/or system forcharacterizing the co-formulated preparations.

SUMMARY

Growth in the development, manufacture and sale of proteinbiopharmaceutical products has led to an increasing demand forcharacterizing the protein biopharmaceutical along with possibleimpurities. Development of stable co-formulated preparations pose anadditional challenge since it requires determination of stability anddegradation of the individual proteins present in the antibody mixture.Such a determination is often difficult due to the large number ofproteins in the formulation, formation of heterodimer species, homodimerspecies and similarities between such proteins.

Exemplary embodiments disclosed herein satisfy the aforementioneddemands by providing methods and/or system for identifying dimer speciesand/or quantifying a heterodimer species.

This disclosure, at least in part, provides a method for identifying adimer species. In one exemplary embodiment, the method for identifying adimer species comprises contacting a sample including the dimer speciesto a chromatographic system with a chromatography resin, washing saidresin using a mobile phase to provide an eluent including the dimerspecies, and identifying the dimer species in said eluent using anelectrospray ionization mass spectrometer.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise a chromatographic system with a size exclusionchromatography resin

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise coupling an electrospray ionization massspectrometer to a chromatographic system with a chromatography resin.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise coupling an electrospray ionization massspectrometer to a chromatographic system having a size-exclusionchromatography resin.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise an electrospray ionization mass spectrometeroperating under native conditions.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise a nano-electrospray ionization mass spectrometer.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise a nano-electrospray ionization mass spectrometeroperating under native conditions.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise at least one splitter with at least three paths tocouple an electrospray ionization mass spectrometer to a chromatographicsystem with a resin.

In one aspect of this embodiment, the method identifying a dimer speciescan comprise at least one splitter with at least three paths to couplean ultraviolet detector to the chromatographic system with a resin.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise at least one splitter with at least three paths tocouple an ultraviolet detector and electrospray ionization massspectrometer to a chromatographic system with a resin.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise at least one splitter with at least three paths tocouple an electrospray ionization mass spectrometer to a chromatographicsystem with a size exclusion chromatography resin.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise at least one splitter with at least three paths tocouple an ultraviolet detector to the chromatographic system with a sizeexclusion chromatography resin.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise at least one splitter with at least three paths tocouple an ultraviolet detector and electrospray ionization massspectrometer to a chromatographic system with a size exclusionchromatography resin.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise washing a resin using the mobile phase to providean eluent including the dimer species, wherein the eluent can beintroduced in an ultraviolet detector through at least one splitter withat least three paths, at a flow rate of about 0.2 mL/min to about 0.4mL/min.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise a mobile phase comprising a volatile salt.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise a mobile phase comprising ammonium acetate.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise washing a resin with the mobile phase with a flowrate of about 0.2 mL/min to about 0.4 mL/min.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise a mobile phase with a pH of about 6.8.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise a sample including the dimer species in an amountof about 10 μg to about 100 μg.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise a dimer species including an antibody.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise a dimer species including a fusion protein.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise an electrospray ionization mass spectrometer with aflow rate of about 10 nL/min to about 50 nL/min.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise an electrospray ionization mass spectrometer with aspray voltage of an electrospray is about 0.8 kV to about 1.5 kV.

In one aspect of this embodiment, the method for identifying a dimerspecies, wherein the dimer species can be a homodimer species.

In one aspect of this embodiment, the method for identifying a dimerspecies, wherein the dimer species can be a heterodimer species.

In one aspect of this embodiment, the method for identifying a dimerspecies can comprise contacting a sample including the dimer species toa chromatographic system with a chromatography resin, wherein the samplecan comprise protein monomers.

This disclosure, at least in part, provides a system comprising achromatographic column having a chromatography resin. In one exemplaryembodiment, the system comprises a chromatographic column having achromatographic resin, wherein the chromatographic column can be capableof receiving a mobile phase and a sample including a protein, and anelectrospray ionization mass spectrometer.

In one aspect of this embodiment, the system can comprise achromatographic column having a size exclusion chromatography resin.

In one aspect of this embodiment, the system can comprise anelectrospray ionization mass spectrometer capable of being coupled tosaid chromatographic column.

In one aspect of this embodiment, the system can comprise anelectrospray ionization mass spectrometer capable of being run undernative conditions.

In one aspect of this embodiment, the system can comprise a nanoelectrospray ionization mass spectrometer.

In one aspect of this embodiment, the system can comprise achromatographic column capable of being coupled to a mass spectrometerusing a splitter with at least three paths.

In one aspect of this embodiment, the system can comprise achromatographic column capable of being coupled to an ultravioletdetector using a splitter with at least three paths.

In one aspect of this embodiment, the system can comprise achromatographic column capable of being coupled to an ultravioletdetector and a mass spectrometer using a splitter with at least threepaths.

In one aspect of this embodiment, the system can be capable ofidentifying a dimer species.

This disclosure, at least in part, provides a method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein, said method comprising immobilizing an antibody specific to thefirst protein on a solid surface, incubating the sample with saidantibody, capturing a precipitated sample, collecting a flow through,treating the precipitated sample with a first compound, treating theflow through with a second compound, mixing the treated precipitatedsample and at least a portion of the treated flow through to form amixture, and analyzing the mixture using a liquid chromatography coupledto a mass spectrometer to quantify the heterodimer species in thesample.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, wherein the first protein can be amonoclonal antibody.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, wherein the solid surface comprises magneticbeads.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, wherein the solid surface comprisesstreptavidin.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface and capturing a precipitated sample, whereinthe precipitated sample includes the heterodimer species bound to theantibody specific to the first protein on a solid surface.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface and capturing a precipitated sample, whereinthe precipitated sample comprises of the heterodimer species bound tothe antibody specific to the first protein on a solid surface.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, capturing a precipitated sample,re-suspending the precipitated sample and heating the re-suspendedprecipitated sample.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, capturing a precipitated sample andcollecting a flow through.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, capturing a precipitated sample andcollecting a flow through, and treating the captured precipitated samplewith a first compound.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, capturing a precipitated sample andcollecting a flow through, and treating the flow through with a firstcompound

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, capturing a precipitated sample andcollecting a flow through, and treating the captured precipitated samplewith a first compound and the flow through with a second compound.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, capturing a precipitated sample andcollecting a flow through, and treating the captured precipitated samplewith a first compound and the flow through with a second compound,wherein the first compound can be an isotope of the second compound.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, capturing a precipitated sample andcollecting a flow through, and treating the captured precipitated samplewith a first compound and the flow through with a second compound, andmixing the treated precipitated sample and at least a portion of thetreated flow through to form a mixture.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, capturing a precipitated sample andcollecting a flow through, and treating the captured precipitated samplewith a first compound and the flow through with a second compound,mixing the treated precipitated sample and at least a portion of thetreated flow through to form a mixture, and digesting the mixture.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, capturing a precipitated sample andcollecting a flow through, and treating the captured precipitated samplewith a first compound and the flow through with a second compound,mixing the treated precipitated sample and at least a portion of thetreated flow through to form a mixture, digesting the mixture, anddeglycosyalting the digested mixture.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, capturing a precipitated sample andcollecting a flow through, and treating the captured precipitated samplewith a first compound and the flow through with a second compound,mixing the treated precipitated sample and at least a portion of thetreated flow through to form a mixture, digesting the mixture, andanalyzing the mixture using a liquid chromatography coupled to a massspectrometer to quantify the heterodimer species in the sample.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, capturing a precipitated sample andcollecting a flow through, and treating the captured precipitated samplewith a first compound and the flow through with a second compound,mixing the treated precipitated sample and at least a portion of thetreated flow through to form a mixture, digesting the mixture,deglycosylating the digested mixture, and analyzing the mixture using aliquid chromatography coupled to a mass spectrometer to quantify theheterodimer species in the sample.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise analyzing using a liquid chromatography coupled toa mass spectrometer to quantify the heterodimer species in the sample,wherein the mass spectrometer can be a tandem mass spectrometer.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, capturing a precipitated sample andcollecting a flow through, and treating the captured precipitated samplewith a first compound and the flow through with a second compound, andadding a reducing agent to the treated precipitated sample.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, capturing a precipitated sample andcollecting a flow through, and treating the captured precipitated samplewith a first compound and the flow through with a second compound, andadding a reducing agent to the treated flow through.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, capturing a precipitated sample andcollecting a flow through, and treating the captured precipitated samplewith a first compound and the flow through with a second compound,mixing the treated precipitated sample and about 10% of the treated flowthrough to form a mixture, and analyzing the mixture using a liquidchromatography coupled to a mass spectrometer to quantify theheterodimer species in the sample.

In one aspect of this embodiment, the method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein can comprise immobilizing an antibody specific to the firstprotein on a solid surface, capturing a precipitated sample andcollecting a flow through, and treating the captured precipitated samplewith a first compound and the flow through with a second compound,mixing the treated precipitated sample and about 10% of the treated flowthrough to form a mixture, digesting the mixture, and analyzing themixture using a liquid chromatography coupled to a mass spectrometer toquantify the heterodimer species in the sample.

This disclosure, at least in part, provides a method for quantifying aheterodimer species. In one exemplary embodiment, the method forquantifying a heterodimer species comprises immunoprecipitating theheterodimer species and quantifying the heterodimer species by using astable isotope labeling method followed by a liquid chromatographycoupled to a mass spectrometer.

In one aspect of this embodiment, the method for quantifying aheterodimer species can comprise a heterodimer species including anantibody.

In one aspect of this embodiment, the method for quantifying aheterodimer species can comprise a heterodimer species including afusion protein.

In one aspect of this embodiment, the method for quantifying aheterodimer species can comprise immunoprecipitating the heterodimerspecies by using an antibody on a solid surface.

In one aspect of this embodiment, the method for quantifying aheterodimer species can comprise immunoprecipitating the heterodimerspecies by using an antibody on a solid surface, wherein the antibodycan bind to a protein from the heterodimer.

In one aspect of this embodiment, the method for quantifying aheterodimer species can comprise using a stable isotope labeling methodwith an alkylating compound.

In one aspect of this embodiment, the method for quantifying aheterodimer species can comprise using a stable isotope labeling methodwith an alkylating compound.

In one aspect of this embodiment, the method for quantifying aheterodimer species can comprise performing digestion in addition tousing a stable isotope labeling method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary embodiment of a system capable of identifyingdimer species.

FIG. 2 shows an exemplary embodiment of the method used to quantifyheterodimer species in a sample of a co-formulated preparation.

FIG. 3 shows a method for quantifying non-specific binding of a proteinto an antibody in a negative sample according to an exemplaryembodiment.

FIG. 4 shows an exemplary embodiment of the method used to quantifyheterodimer species in a sample of a co-formulated preparation.

FIG. 5 shows the six dimers which may possibly be present in aco-formulation comprising three monoclonal antibodies.

FIG. 6 shows identification of heterodimer species in a co-formulationcomprising three monoclonal antibodies using native SEC-MS analysisaccording to an exemplary embodiment.

FIG. 7 shows quantification of heterodimer species in a co-formulationcomprising three monoclonal antibodies using native SEC-MS analysisaccording to an exemplary embodiment.

FIG. 8 shows the three dimers which may possibly be present in aco-formulation comprising a monoclonal antibody and a fusion protein.

FIG. 9 shows an extracted ion chromatogram (XIC) of a co-formulationdifferentiated according to an exemplary embodiment.

FIG. 10 shows mass to charge ratio determination of dimer species of aco-formulation differentiated according to an exemplary embodiment.

FIG. 11 shows a workflow of an immunoprecipitation and isotope-labelingstrategy for heterodimer quantitation according to an exemplaryembodiment.

FIG. 12 shows the mass to charge ratios of the pull-down andflow-through fractions obtained for cool white stressed co-formulationand a negative sample according to an exemplary embodiment.

FIG. 13 shows a chart for fusion protein 1 heterodimer % in fourpeptides of fusion protein 1 found in samples stressed under differentconditions, wherein the quantitation was performed according to anexemplary embodiment.

FIG. 14 shows chart of mass to charge ratio of fusion protein 1 peptide1 (both light and heavy fragments) in samples stressed under differentconditions, wherein the quantitation of a heterodimer comprising fusionprotein 1 was performed according to an exemplary embodiment.

FIG. 15 shows chart of mass to charge ratio of fusion protein 1 peptide2 (both light and heavy fragments) in samples stressed under differentconditions, wherein the quantitation of a heterodimer comprising fusionprotein 1 was performed according to an exemplary embodiment.

FIG. 16 shows chart of mass to charge ratio of fusion protein 1 peptide3 (both light and heavy fragments) in samples stressed under differentconditions, wherein the quantitation of a heterodimer comprising fusionprotein 1 was performed according to an exemplary embodiment.

FIG. 17 shows chart of mass to charge ratio of fusion protein 1 peptide4 (both light and heavy fragments) in samples stressed under differentconditions, wherein the quantitation of a heterodimer comprising fusionprotein 1 was performed according to an exemplary embodiment.

FIG. 18 shows the calculations for converting fusion protein 1heterodimer % to heterodimer %, wherein the quantitation of aheterodimer comprising fusion protein 1 was performed according to anexemplary embodiment.

FIG. 19 represents a chart of fusion protein 1 heterodimer % in thetested samples normalized to fusion protein heterodimer % in the testedsample with no dilution, wherein the quantitation of a heterodimercomprising fusion protein 1 was performed according to an exemplaryembodiment.

FIG. 20 shows a plot of fusion protein 1 heterodimer % vs. % cool-whitestressed sample in the mixed sample for three of peptides used toquantify fusion protein 1, wherein the quantitation of a heterodimercomprising fusion protein 1 was performed according to an exemplaryembodiment.

FIG. 21 shows the fusion protein 1 heterodimer % in samples fromco-formulated preparations stored at ambient conditions, wherein thequantitation of a heterodimer comprising fusion protein 1 was performedaccording to an exemplary embodiment.

DETAILED DESCRIPTION

Identification and quantification of proteins in proteinbiopharmaceutical products may be very important during the productionand development of a product. The analysis of impurities in any proteinbiopharmaceutical product may be imperative into developing a safe andeffective product. Hence, a robust method and/or workflow tocharacterize the impurities may be beneficial.

Most biopharmaceutical products may comprise a single protein. In someinstances, multiple proteins directed to a single target or multipletargets administered in combination may improve their diagnostic ortherapeutic indication and efficacy. Development of such co-formulatedpreparations are becoming increasingly popular dosage forms (SvendHavelund et al., Investigation of the Physico-Chemical Properties thatEnable Co-Formulation of Basal Insulin Degludec with Fast-Acting InsulinAspart, 32 PHARMACEUTICAL RESEARCH 2250-2258 (2015); Sanjay Kalra &Yashdeep Gupta, Injectable Coformulations in Diabetology, 6 DIABETESTHERAPY101-111 (2015); Ryzodeg, EUROPEAN MEDICINES AGENCY-FINDMEDICINE-RAXONE,https://www.ema.europa.eu/en/medicines/human/EPAR/ryzodeg (last visitedJan. 17, 2019)).

The development of co-formulated preparations, however, has somechallenges, since it requires studying the potential pharmacodynamicsinteractions, potential pharmacokinetic interactions, potential fortoxicological interactions, potential for changes in the levels oractivity of endogenous, and potential for impairing the efficacy of oneof the drugs (Claudia Mueller, Ulrike Altenburger & Silke Mohl,Challenges for the pharmaceutical technical development of proteincoformulations, 70 JOURNAL OF PHARMACY AND PHARMACOLOGY 666-674 (2017)).The manufacture of stable co-formulated preparation may also requireadditional efforts to ensure the stability of the two more proteins atvarious steps of the manufacturing.

During storage, transport and administration, the final co-formulatedproducts may get exposed to light, heat and oxygen and cause aggregation(high molecular weight species homodimer(s) and/or heterodimer(s)),charge pattern, fragmentation, chemical modifications (e.g. oxidation,deamidation), etc.

A good analytical method performance and sensitivity are crucial toensure high quality and safety of the final co-formulated product.However, the analytical approach for the development and evaluation ofco-formulated preparations may be significantly challenging and complexthan approach applied for formulations comprising a single protein as anactive pharmaceutical ingredient. This may become challenging as certainstandard methods employed for a formulation comprising a single proteinmay not always be capable of differentiating proteins in theco-formulated preparations.

One of the methods includes use of size exclusion chromatography (SEC)for characterizing biomolecular aggregation and fragmentation in thebiotech industry (Hong Paule et al., Size-Exclusion Chromatography forthe Analysis of Protein Biotherapeutics and their Aggregates, 35 JOURNALOF LIQUID CHROMATOGRAPHY AND RELATED TECHNOLOGY 2923-2950 (2012)). Theseparation of molecules by SEC relies on the differential interaction ofmolecules with a controlled porous structure on a stationary phase. AsSEC uses buffer conditions that preserve the native structure ofproteins in solution, it permits characterization of biomoleculeswithout disturbing their native conformation. Further, among the variousdetection modes that may be coupled with SEC, mass spectrometry (MS)allows for the precise and accurate identification of individualcomponents in complex samples. The combination of SEC and MS has beenreported previously, including the collection of SEC peaks followed bydirect infusion MS (Başak Kükrer et al., Mass Spectrometric Analysis ofIntact Human Monoclonal Antibody Aggregates Fractionated bySize-Exclusion Chromatography, 27 PHARMACEUTICAL RESEARCH 2197-2204(2010); François Debaene et al., Innovative Native MS Methodologies forAntibody Drug Conjugate Characterization: High Resolution Native MS andIM-MS for Average DAR and DAR Distribution Assessment, 86 ANALYTICALCHEMISTRY 10674-10683 (2014)) or online SEC-MS (Khaja Muneeruddin etal., Characterization of Small Protein Aggregates and Oligomers UsingSize Exclusion Chromatography with Online Detection by NativeElectrospray Ionization Mass Spectrometry, 86 ANALYTICAL CHEMISTRY10692-10699 (2014); C. F. Mcdonagh et al., Engineered antibody- drugconjugates with defined sites and stoichiometries of drug attachment, 19PROTEIN ENGINEERING DESIGN AND SELECTION 299-307 (2006)). However, todirectly ionize the high flow generated from SEC separation requiresharsh ionization conditions that are often incompatible with native MSanalysis, thereby limiting the utility of coupling these technologiesfor the analysis of non-covalent interactions. Further, the sensitivityof the mass spectrometer may suffer from the high salt concentrationsused in SEC buffers.

In addition to the identification, quantification of heterodimer(s)formed in the co-formulated product may also raise challenges, since theamount of heterodimer(s) formed needs to evaluated in presence of theprotein monomers and any present homodimer(s).

Considering the limitations of existing methods, effective and efficientmethods for identification and quantification of dimer species wasdeveloped.

Unless described otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein may beused in the practice or testing, particular methods and materials arenow described. All publications mentioned are hereby incorporated byreference.

The term “a” should be understood to mean “at least one”; and the terms“about” and “approximately” should be understood to permit standardvariation as would be understood by those of ordinary skill in the art;and where ranges are provided, endpoints are included.

In some exemplary embodiments, the disclosure provides a method foridentifying dimer species in a co-formulated preparation.

As used herein, a “co-formulated preparation” includes two or moreactive pharmaceutical ingredients in a single dosage form. This dosageform can be used to treat, prevent, or ameliorate a certain diseasecondition by targeting different molecular targets and obtain an overallimproved medical condition of the patient due to additive and/orsynergistic effects as compared to the single dug(s) alone (ClaudiaMueller, Ulrike Altenburger & Silke Mohl, Challenges for thepharmaceutical technical development of protein coformulations, 70JOURNAL OF PHARMACY AND PHARMACOLOGY 666-674 (2017)). Some of theadvantages include increased efficacy compared to a single drug, overallreduction of adverse event, improvement of patient convenience andcompliance (increased patient adherence, simplified patient guidance andeducation), reduction in health care costs (manufacture and purchase),easier supply processes, and new product opportunities within life-cyclemanagement of existing marketed products.

In some exemplary embodiments, the two or more active pharmaceuticalingredients can be proteins.

In some exemplary embodiments, the co-formulated preparation cancomprise at least two proteins.

In some exemplary embodiments, the co-formulated preparation cancomprise at least three proteins.

As used herein, the term “protein” includes any amino acid polymerhaving covalently linked amide bonds. Proteins comprise one or moreamino acid polymer chains, generally known in the art as “polypeptides”.“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. “Synthetic peptides orpolypeptides' refers to a non-naturally occurring peptide orpolypeptide. Synthetic peptides or polypeptides can be synthesized, forexample, using an automated polypeptide synthesizer. Various solid phasepeptide synthesis methods are known. A protein may contain one ormultiple polypeptides to form a single functioning biomolecule. Aprotein can include any of bio-therapeutic proteins, recombinantproteins used in research or therapy, trap proteins and other chimericreceptor Fc-fusion proteins, chimeric proteins, antibodies, monoclonalantibodies, polyclonal antibodies, human antibodies, and bispecificantibodies. In another exemplary aspect, a protein can include antibodyfragments, nanobodies, recombinant antibody chimeras, cytokines,chemokines, peptide hormones, and the like. Proteins may be producedusing recombinant cell-based production systems, such as the insectbacculovirus system, yeast systems (e.g., Pichia sp.), mammalian systems(e.g., CHO cells and CHO derivatives like CHO-K1 cells). For a reviewdiscussing biotherapeutic proteins and their production, see Ghaderi etal., “Production platforms for biotherapeutic glycoproteins. Occurrence,impact, and challenges of non-human sialylation,” (BIOTECHNOL. GENET.ENG. REV. 147-175 (2012)). In some exemplary embodiments, proteinscomprise modifications, adducts, and other covalently linked moieties.Those modifications, adducts and moieties include for example avidin,streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose,neuraminic acid, N-acetylglucosamine, fucose, mannose, and othermonosaccharides), PEG, polyhistidine, FLAGtag, maltose binding protein(MBP), chitin binding protein (CBP), glutathione-S-transferase (GST)myc-epitope, fluorescent labels and other dyes, and the like. Proteinscan be classified on the basis of compositions and solubility and canthus include simple proteins, such as, globular proteins and fibrousproteins; conjugated proteins, such as nucleoproteins, glycoproteins,mucoproteins, chromoproteins, phosphoproteins, metalloproteins, andlipoproteins; and derived proteins, such as primary derived proteins andsecondary derived proteins.

In some exemplary embodiments, the protein can be an antibody, abispecific antibody, a multispecific antibody, antibody fragment,monoclonal antibody, or an Fc fusion protein. In one aspect, the proteinis a anti-VEGF protein. In a specific aspect, the VEGF-protein isAflibercept.

The term “antibody,” as used herein includes immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds, as well as multimersthereof (e.g., IgM). Each heavy chain comprises a heavy chain variableregion (abbreviated herein as HCVR or V_(H)) and a heavy chain constantregion. The heavy chain constant region comprises three domains, C_(H)1,C_(H)2 and C_(H)3. Each light chain comprises a light chain variableregion (abbreviated herein as LCVR or VL) and a light chain constantregion. The light chain constant region comprises one domain (Cu). TheV_(H) and VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different exemplaryembodiments, the FRs of the anti-big-ET-1 antibody (or antigen-bindingportion thereof) may be identical to the human germline sequences, ormay be naturally or artificially modified. An amino acid consensussequence may be defined based on a side-by-side analysis of two or moreCDRs. The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

As used herein, an “antibody fragment” includes a portion of an intactantibody, such as, for example, the antigen-binding or variable regionof an antibody. Examples of antibody fragments include, but are notlimited to, a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a Fcfragment, a scFv fragment, a Fv fragment, a dsFv diabody, a dAbfragment, a Fd′ fragment, a Fd fragment, and an isolated complementaritydetermining region (CDR) region, as well as triabodies, tetrabodies,linear antibodies, single-chain antibody molecules, and multi specificantibodies formed from antibody fragments. Fv fragments are thecombination of the variable regions of the immunoglobulin heavy andlight chains, and ScFv proteins are recombinant single chain polypeptidemolecules in which immunoglobulin light and heavy chain variable regionsare connected by a peptide linker. An antibody fragment may be producedby various means. For example, an antibody fragment may be enzymaticallyor chemically produced by fragmentation of an intact antibody and/or itmay be recombinantly produced from a gene encoding the partial antibodysequence. Alternatively or additionally, an antibody fragment may bewholly or partially synthetically produced. An antibody fragment mayoptionally comprise a single chain antibody fragment. Alternatively oradditionally, an antibody fragment may comprise multiple chains that arelinked together, for example, by disulfide linkages. An antibodyfragment may optionally comprise a multi-molecular complex.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. A monoclonal antibodycan be derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, by any means available or known in the art.Monoclonal antibodies useful with the present disclosure can be preparedusing a wide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof.

The term “Fc fusion proteins” as used herein includes part or all of twoor more proteins, one of which is an Fc portion of an immunoglobulinmolecule, that are not fused in their natural state. Preparation offusion proteins comprising certain heterologous polypeptides fused tovarious portions of antibody-derived polypeptides (including the Fcdomain) has been described, e.g., by Ashkenazi et al., Proc. Natl. Acad.ScL USA 88: 10535, 1991; Byrn et al., Nature 344:677, 1990; andHollenbaugh et al., “Construction of Immunoglobulin Fusion Proteins”, inCurrent Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992.“Receptor Fc fusion proteins” comprise one or more of one or moreextracellular domain(s) of a receptor coupled to an Fc moiety, which insome embodiments comprises a hinge region followed by a CH2 and CH3domain of an immunoglobulin. In some embodiments, the Fc-fusion proteincontains two or more distinct receptor chains that bind to a single ormore than one ligand(s). For example, an Fc-fusion protein is a trap,such as for example an IL-1 trap (e.g., Rilonacept, which contains theIL-1 RAcP ligand binding region fused to the IL-1R1 extracellular regionfused to Fc of hIgG1; see U.S. Pat. No. 6,927,004, which is hereinincorporated by reference in its entirety), or a VEGF Trap (e.g.,Aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1fused to the Ig domain 3 of the VEGF receptor Flk1 fused to Fc of hIgG1;e.g., SEQ ID NO:1; see U.S. Pat. Nos. 7,087,411 and 7,279,159, which areherein incorporated by reference in their entirety)

In some exemplary embodiments, the co-formulated preparation cancomprise dimer species. In one aspect, the co-formulated preparation cancomprise homodimer species. In some other specific exemplaryembodiments, the co-formulated preparation can comprise heterodimerspecies. In another aspect, the co-formulated preparation can comprisehomodimer species and heterodimer species.

In some exemplary embodiments, the co-formulated preparation cancomprise dimer species as an impurity.

As used herein, the term “impurity” can include any undesirable proteinpresent in the protein biopharmaceutical product. Impurity can includeprocess and product-related impurities. The impurity can further be ofknown structure, partially characterized, or unidentified.Process-related impurities can be derived from the manufacturing processand can include the three major categories: cell substrate-derived, cellculture-derived and downstream derived. Cell substrate-derivedimpurities include, but are not limited to, proteins derived from thehost organism and nucleic acid (host cell genomic, vector, or totalDNA). Cell culture-derived impurities include, but are not limited to,inducers, antibiotics, serum, and other media components.Downstream-derived impurities include, but are not limited to, enzymes,chemical and biochemical processing reagents (e.g., cyanogen bromide,guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavymetals, arsenic, nonmetallic ion), solvents, carriers, ligands (e.g.,monoclonal antibodies), and other leachables. Product-related impurities(e.g., precursors, certain degradation products) can be molecularvariants arising during manufacture and/or storage that do not haveproperties comparable to those of the desired product with respect toactivity, efficacy, and safety. Such variants may need considerableeffort in isolation and characterization in order to identify the typeof modification(s). Product-related impurities can include truncatedforms, modified forms, and aggregates. Truncated forms are formed byhydrolytic enzymes or chemicals which catalyze the cleavage of peptidebonds. Modified forms include, but are not limited to, deamidated;isomerized, mismatched S—S linked, oxidized, or altered conjugated forms(e.g., glycosylation, phosphorylation). Modified forms can also includeany post-translational modification form. Aggregates include dimers andhigher multiples of the desired product. (Q6B Specifications: TestProcedures and Acceptance Criteria for Biotechnological/BiologicalProducts, ICH August 1999, U.S. Dept. of Health and Humans Services).

As used herein, the general term “post-translational modifications” or“PTMs” refer to covalent modifications that polypeptides undergo, eitherduring (co-translational modification) or after (post-translationalmodification) their ribosomal synthesis. PTMs are generally introducedby specific enzymes or enzyme pathways. Many occur at the site of aspecific characteristic protein sequence (signature sequence) within theprotein backbone. Several hundred PTMs have been recorded, and thesemodifications invariably influence some aspect of a protein's structureor function (Walsh, G. “Proteins” (2014) second edition, published byWiley and Sons, Ltd., ISBN: 9780470669853). The variouspost-translational modifications include, but are not limited to,cleavage, N-terminal extensions, protein degradation, acylation of theN-terminus, biotinylation (acylation of lysine residues with a biotin),amidation of the C-terminal, glycosylation, iodination, covalentattachment of prosthetic groups, acetylation (the addition of an acetylgroup, usually at the N-terminus of the protein), alkylation (theaddition of an alkyl group (e.g. methyl, ethyl, propyl) usually atlysine or arginine residues), methylation, adenylation,ADP-ribosylation, covalent cross links within, or between, polypeptidechains, sulfonation, prenylation, Vitamin C dependent modifications(proline and lysine hydroxylations and carboxy terminal amidation),Vitamin K dependent modification wherein Vitamin K is a cofactor in thecarboxylation of glutamic acid residues resulting in the formation of aγ-carboxyglutamate (a glu residue), glutamylation (covalent linkage ofglutamic acid residues), glycylation (covalent linkage glycineresidues), glycosylation (addition of a glycosyl group to eitherasparagine, hydroxylysine, serine, or threonine, resulting in aglycoprotein), isoprenylation (addition of an isoprenoid group such asfarnesol and geranylgeraniol), lipoylation (attachment of a lipoatefunctionality), phosphopantetheinylation (addition of a4′-phosphopantetheinyl moiety from coenzyme A, as in fatty acid,polyketide, non-ribosomal peptide and leucine biosynthesis),phosphorylation (addition of a phosphate group, usually to serine,tyrosine, threonine or histidine), and sulfation (addition of a sulfategroup, usually to a tyrosine residue). The post-translationalmodifications that change the chemical nature of amino acids include,but are not limited to, citrullination (the conversion of arginine tocitrulline by deimination), and deamidation (the conversion of glutamineto glutamic acid or asparagine to aspartic acid). The post-translationalmodifications that involve structural changes include, but are notlimited to, formation of disulfide bridges (covalent linkage of twocysteine amino acids) and proteolytic cleavage (cleavage of a protein ata peptide bond). Certain post-translational modifications involve theaddition of other proteins or peptides, such as ISGylation (covalentlinkage to the ISG15 protein (Interferon-Stimulated Gene)), SUMOylation(covalent linkage to the SUMO protein (Small Ubiquitin-relatedMOdifier)) and ubiquitination (covalent linkage to the proteinubiquitin). See European Bioinformatics Institute Protein InformationResourceSlB Swiss Institute of Bioinformatics, EUROPEAN BIOINFORMATICSINSTITUTE DRS-DROSOMYCIN PRECURSOR-DROSOPHILA MELANOGASTER (FRUITFLY)-DRS GENE & PROTEIN, http://www.uniprot.org/docs/ptmlist (lastvisited Jan. 15, 2019) for a more detailed controlled vocabulary of PTMscurated by UniProt.

In some exemplary embodiments, dimer species can be identified bycontacting a sample including the dimer species to a chromatographicsystem.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas can be separated intocomponents as a result of differential distribution of the chemicalentities as they flow around or over a stationary liquid or solid phase.Non-limiting examples of chromatography include traditionalreversed-phased (RP), ion exchange (IEX), mixed mode chromatography andnormal phase chromatography (NP).

As used herein, the term “Mixed Mode Chromatography (MMC)” or“multimodal chromatography” includes a chromatographic method in whichsolutes interact with stationary phase through more than one interactionmode or mechanism. MMC can be used as an alternative or complementarytool to traditional reversed-phased (RP), ion exchange (IEX) and normalphase chromatography (NP). Unlike RP, NP and IEX chromatography, inwhich hydrophobic interaction, hydrophilic interaction and ionicinteraction respectively are the dominant interaction modes, mixed-modechromatography can employ a combination of two or more of theseinteraction modes. Mixed mode chromatography media can provide uniqueselectivity that cannot be reproduced by single mode chromatography.Mixed mode chromatography can also provide potential cost savings andoperation flexibility compared to affinity based methods.

In some exemplary embodiments, the chromatography can be size-exclusionchromatography.

As used herein, the terms “SEC chromatography resin” or “SECchromatography media” are used interchangeably and can include any kindof solid phase used in SEC which separates the impurity from the desiredproduct (e.g., a homodimer contaminant for a bispecific antibodyproduct). The volume of the resin, the length and diameter of the columnto be used, as well as the dynamic capacity and flow-rate can depend onseveral parameters such as the volume of fluid to be treated,concentration of protein in the fluid to be subjected to the process.

In some exemplary embodiments, the method for identifying dimer speciescan comprise contacting a sample including the protein biopharmaceuticalto a chromatographic system using a mobile phase to provide an eluentincluding the dimer species; and identifying the protein in said eluentusing an electrospray ionization mass spectrometer.

As used herein, the term “mass spectrometer” includes a device capableof identifying specific molecular species and measuring their accuratemasses. The term is meant to include any molecular detector into which apolypeptide or peptide may be eluted for detection and/orcharacterization. A mass spectrometer can include three major parts: theion source, the mass analyzer, and the detector. The role of the ionsource is to create gas phase ions. Analyte atoms, molecules, orclusters can be transferred into gas phase and ionized eitherconcurrently (as in electrospray ionization). The choice of ion sourcedepends heavily on the application.

In some embodiments, the mass spectrometer can be an electrospray-massspectrometer.

As used herein, the term “electrospray ionization” or “ESI” refers tothe process of spray ionization in which either cations or anions insolution are transferred to the gas phase via formation and desolvationat atmospheric pressure of a stream of highly charged droplets thatresult from applying a potential difference between the tip of theelectrospray needle containing the solution and a counter electrode.There are generally three major steps in the production of gas-phaseions from electrolyte ions in solution. These are: (a) production ofcharged droplets at the ES infusion tip; (b) shrinkage of chargeddroplets by solvent evaporation and repeated droplet disintegrationsleading to small highly charged droplets capable of producing gas-phaseions; and (c) the mechanism by which gas-phase ions are produced fromvery small and highly charged droplets. Stages (a)-(c) generally occurin the atmospheric pressure region of the apparatus.

As used herein, the term “electrospray infusion setup” refers to anelectrospray ionization system that is compatible with a massspectrometer used for mass analysis of protein. In electrosprayionization, an electrospray needle has its orifice positioned close tothe entrance orifice of a spectrometer. A sample, containing the proteinof interest, can be pumped through the syringe needle. An electricpotential between the syringe needle orifice and an orifice leading tothe mass analyzer forms a spray (“electrospray”) of the solution. Theelectrospray can be carried out at atmospheric pressure and provideshighly charged droplets of the solution. The electrospray infusion setupcan include an electrospray emitter, nebulization gas, and/or an ESIpower supply. The setup can optionally be automated to carry out sampleaspiration, sample dispensing, sample delivery, and/or for spraying thesample.

In some exemplary embodiments, the electrospray ionization massspectrometer can be a nano-electrospray ionization mass spectrometer.

The term “nanoelectrospray” or “nanospray” as used herein refers toelectrospray ionization at a very low solvent flow rate, typicallyhundreds of nanoliters per minute of sample solution or lower, oftenwithout the use of an external solvent delivery. The electrosprayinfusion setup forming a nanoelectrospray can use a staticnanoelectrospray emitter or a dynamic nanoelectrospray emitter. A staticnanoelectrospray emitter performs a continuous analysis of small sample(analyte) solution volumes over an extended period of time. A dynamicnanoelectrospray emitter uses a capillary column and a solvent deliverysystem to perform chromatographic separations on mixtures prior toanalysis by the mass spectrometer.

As used herein, the term “mass analyzer” includes a device that canseparate species, that is, atoms, molecules, or clusters, according totheir mass. Non-limiting examples of mass analyzers that could beemployed for fast protein sequencing are time-of-flight (TOF),magnetic/electric sector, quadrupole mass filter (Q), quadrupole iontrap (QIT), orbitrap, Fourier transform ion cyclotron resonance (FTICR),and also the technique of accelerator mass spectrometry (AMS).

In some exemplary embodiments, mass spectrometry can be performed undernative conditions.

As used herein, the term “native conditions” or “native MS” or “nativeESI-MS” can include a performing mass spectrometry under conditions thatpreserve no-covalent interactions in an analyte. For detailed review onnative MS, refer to the review: Elisabetta Boeri Erba & Carlo Petosa,The emerging role of native mass spectrometry in characterizing thestructure and dynamics of macromolecular complexes, 24 PROTEIN SCIENCE1176-1192 (2015). Some of the distinctions between native ESI andregular ESI are illustrated in table 1 (Hao Zhang et al., Native massspectrometry of photosynthetic pigment-protein complexes, 587 FEBSLetters 1012-1020 (2013)).

TABLE 1 Native ESI Regular ESI Sample Aqueous solution Partial organicsolution Solution water, ammonium water, formic acid, acetateacetonitrile/Methanol (pH 1-2) Spray 10-50 nL/min 10-50 nL/min ConditionSpray voltage 0.8-1.5 kV Spray voltage 0.8-1.5 kV Temperatures 20-30° C.Temperatures 20-30° C. Salt Treatment Offline Desalt Online/OfflineDesalt with RP-HPLC Protein 1-10 μM (complex) <1 μM (subunit)Concentration Output Molecular weight of Molecular weight of aInformation protein complex and single subunit subunit Non-covalentinteractions Stoichiometry Structure

In some exemplary embodiments, the mass spectrometer can be a tandemmass spectrometer.

As used herein, the term “tandem mass spectrometry” includes a techniquewhere structural information on sample molecules is obtained by usingmultiple stages of mass selection and mass separation. A prerequisite isthat the sample molecules can be transferred into gas phase and ionizedintact and that they can be induced to fall apart in some predictableand controllable fashion after the first mass selection step. MultistageMS/MS, or MS^(n), can be performed by first selecting and isolating aprecursor ion (MS²), fragmenting it, isolating a primary fragment ion(MS³), fragmenting it, isolating a secondary fragment (MS⁴), and so onas long as one can obtain meaningful information or the fragment ionsignal is detectable. Tandem MS have been successfully performed with awide variety of analyzer combinations. What analyzers to combine for acertain application is determined by many different factors, such assensitivity, selectivity, and speed, but also size, cost, andavailability. The two major categories of tandem MS methods aretandem-in-space and tandem-in-time, but there are also hybrids wheretandem-in-time analyzers are coupled in space or with tandem-in-spaceanalyzers. A tandem-in-space mass spectrometer comprises an ion source,a precursor ion activation device, and at least two non-trapping massanalyzers. Specific m/z separation functions can be designed so that inone section of the instrument ions are selected, dissociated in anintermediate region, and the product ions are then transmitted toanother analyzer for m/z separation and data acquisition. Intandem-in-time mass spectrometer ions produced in the ion source can betrapped, isolated, fragmented, and m/z separated in the same physicaldevice.

The peptides identified by the mass spectrometer can be used assurrogate representatives of the intact protein and theirpost-translational modifications. They can be used for proteincharacterization by correlating experimental and theoretical MS/MS data,the latter generated from possible peptides in a protein sequencedatabase. The characterization can include, but is not limited, tosequencing amino acids of the protein fragments, determining proteinsequencing, determining protein de novo sequencing, locatingpost-translational modifications, or identifying post translationalmodifications, or comparability analysis, or combinations thereof.

As used herein, the term “database” refers to bioinformatic tools whichprovide the possibility of searching the uninterpreted MS-MS spectraagainst all possible sequences in the database(s). Non-limiting examplesof such tools are Mascot (http://www.matrixscience.com), Spectrum Mill(http://www.chem.agilent.com), PLGS (http://www.waters.com), PEAKS(http://www.bioinformaticssolutions.com), Proteinpilot(http://download.appliedbiosystems.com//proteinpilot), Phenyx(http://www.phenyx-ms.com), Sorcerer(http://www.sagenresearch.com), OMSSA (http://www.pubchem.ncbi.nlm.nih.gov/omssa/), X!Tandem(http://www.thegpm.org/TANDEM/), Protein Prospector (http://www.http://prospector.ucsf.edu/prospector/mshome.htm), Byonic(https://www.proteinmetrics.com/products/byonic) or Sequest(http://fields.scripps.edu/sequest).

In some exemplary embodiments, the disclosure provides a method forquantifying a heterodimer species.

In some embodiments, the method for quantifying a heterodimer species ina comprising a first protein and a second protein can compriseimmobilizing an antibody specific to the first protein on a solidsurface. The antibody specific to the first protein can be preparedusing any of the known methods in the literature.

As used herein, the term “solid surface” can include any surface with anability to bind to an antibody of interest. In some embodiments for asample comprising a first protein and a second protein, the antibody ofinterest can be an antibody specific to the first protein. Non-limitingexamples of solid surface can include affinity resins, magnetic beadsand coated plates with an immobilized protein, such as, avidin,streptavidin, or NeutrAvidin.

In some embodiments, the method for quantifying a heterodimer speciescan comprise immunoprecipitating the heterodimer species. Theimmunoprecipitation can be performed by using an antibody immobilized ona solid surface, wherein the antibody can bind to one of many activepharmaceutical ingredients in co-formulated preparation, and wherein theactive pharmaceutical ingredients can be proteins.

In some embodiments, the sample comprising a first protein and a secondprotein can be incubated with an immobilized antibody specific to thefirst protein on a solid surface. The time of incubation can range fromseveral minutes to several hours.

In some embodiments, the sample comprising a first protein and a secondprotein incubated with an immobilized antibody specific to the firstprotein on a solid surface can be pulled down.

In some embodiments, the sample comprising a first protein and a secondprotein incubated with an immobilized antibody specific to the firstprotein on a solid surface can be centrifuged. In some specificembodiments, the centrifugation can produce a precipitated sample and aflow through or supernatant.

In some embodiments, the precipitated sample and the flow through can bereduced by using a reducing agent.

As used herein, the term “reducing” refers to the reduction of disulfidebridges in a protein. Non-limiting examples of the reducing agents usedto reduce the protein are dithiothreitol (DTT), β-mercaptoethanol,Ellman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride,tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or combinationsthereof.

In some embodiments, the precipitated sample and/or flow through can betreated using a compound or compounds. In some specific embodiments, thetreatment can include alkylation. In some other specific exemplaryembodiments, the treatment can include alkylation of sulfhydryl groupson a protein.

As used herein, the term “treating” or “isotopically labeling” can referto chemical labeling a protein. Non-selected examples of the methods tochemical labeling a protein include Isobaric tags for relative andabsolute quantitation (iTRAQ) using reagents, such as 4-plex, 6-plex,and 8-plex; reductive demethylation of amines, carbamylation of amines,¹⁸O-labeling on the C-terminus of the protein, or any amine- orsulfhydryl-group of the protein to label amines or sulfhydryl group.

In some embodiments, the precipitated sample and/or flow through can betreated using two distinct compounds, wherein the two compounds areisotopes.

In some embodiments, the precipitated sample and at least a portion ofthe flow through can be mixed after treating or isotopically labelingthem.

In some embodiments, the mixture of the treated or isotopically labeledprecipitated sample and at least a portion of the treated orisotopically labeled flow through can be digested.

As used herein, the term “digestion” refers to hydrolysis of one or morepeptide bonds of a protein. There are several approaches to carrying outdigestion of a protein in a sample using an appropriate hydrolyzingagent, for example, enzymatic digestion or non-enzymatic digestion.

As used herein, the term “hydrolyzing agent” refers to any one orcombination of a large number of different agents that can performdigestion of a protein. Non-limiting examples of hydrolyzing agents thatcan carry out enzymatic digestion include trypsin, endoproteinase Arg-C,endoproteinase Asp-N, endoproteinase Glu-C, outer membrane protease T(OmpT), immunoglobulin-degrading enzyme of Streptococcus pyogenes(IdeS), chymotrypsin, pepsin, thermolysin, papain, pronase, and proteasefrom Aspergillus saitoi. Non-limiting examples of hydrolyzing agentsthat can carry out non-enzymatic digestion include the use of hightemperature, microwave, ultrasound, high pressure, infrared, solvents(non-limiting examples are ethanol and acetonitrile), immobilized enzymedigestion (IMER), magnetic particle immobilized enzymes, and on-chipimmobilized enzymes. For a recent review discussing the availabletechniques for protein digestion see Switazar et al., “ProteinDigestion: An Overview of the Available Techniques and RecentDevelopments” (J. Proteome Research 2013, 12, 1067-1077). One or acombination of hydrolyzing agents can cleave peptide bonds in a proteinor polypeptide, in a sequence-specific manner, generating a predictablecollection of shorter peptides.

In some embodiments, on digestion of mixture of the treated orisotopically labeled precipitated sample and at least a portion of thetreated or isotopically labeled flow through can be analyzed using aliquid chromatography coupled to a mass spectrometer. One such exemplaryembodiment is represented on FIG. 2 . FIG. 2 shows an exemplaryembodiment for a method for quantifying a heterodimer species in asample comprising a first protein—mAb1 and a second protein—fusionprotein 1 in a co-formulated preparation, said method comprisingimmobilizing an antibody specific to the first protein (anti-mAb1antibody) on a solid surface, incubating the sample with said antibody,capturing a precipitated sample by pulling down the heterodimer of mAb1and fusion protein 1, mAb1 monomer and mAb1 dimer, collecting a flowthrough comprising fusion protein 1 monomer and fusion protein 1 dimer,treating the precipitated sample with a first compound—unlabelediodoacetamide (IAA), treating the flow through with a secondcompound—labeled iodoacetamide (IAA), mixing the treated precipitatedsample and about 10% of the treated flow through to form a mixture,digesting the said mixture, and analyzing the mixture using a liquidchromatography coupled to a mass spectrometer to quantify theheterodimer species in the sample. During quantitation of theheterodimer species using an exemplary embodiment, non-specific bindingof the second protein on incubating the sample with said antibody. Sucha non-specific binding could lead to overestimation of the quantity ofthe heterodimer can occur. Hence, a negative sample can be prepared andthe amount of the non-specifically bound second protein can be obtained.An example of one such possibility is shown in FIG. 3 .

In some exemplary embodiments, the negative sample can comprise monomersand homodimers of a first protein and a second protein, without thepresence of a heterodimer species.

FIG. 3 shows an exemplary embodiment for preparing a negative sample. Itcomprises using a sample comprising monomers and homodimers of a firstprotein (mAb1) and a second protein (fusion protein 1). This amount ofsecond protein non-specifically bound to the antibody can be quantifiedby using a method comprising immobilizing an antibody specific to thefirst protein (anti-mAb1 antibody) on a solid surface, incubating thesample with said antibody, capturing a precipitated sample by pullingdown non-specifically bound fusion protein 1, mAb1 monomer and mAb1dimer, collecting a flow through comprising fusion protein 1 monomer anddimer, treating the precipitated sample with a first compound—unlabelediodoacetamide (IAA), treating the flow through with a secondcompound—labeled iodoacetamide (IAA), mixing the treated precipitatedsample and about 10% of the treated flow through to form a mixture, andanalyzing the mixture using a liquid chromatography coupled to a massspectrometer to quantify the heterodimer species in the sample.

Exemplary Embodiments

Embodiments disclosed herein provide methods and system for theidentification and/or quantification of dimer species.

In some exemplary embodiments, this disclosure provides a method foridentification of a dimer species, comprising contacting a sampleincluding the dimer species to a chromatographic system with achromatography resin, washing said resin using a mobile phase to providean eluent including the dimer species, and identifying the dimer speciesin said eluent using an electrospray ionization mass spectrometer.

In some exemplary embodiments, dimer species can be a homodimer species.

In some exemplary embodiments, dimer species can be a heterodimerspecies.

In some exemplary embodiments, the chromatographic system can includetraditional reversed-phased (RP), ion exchange (IEX) or normal phasechromatography (NP).

In some exemplary embodiments, the chromatographic resin can be selectedfrom affinity chromatography resin, anion-exchange resin, cationexchange resin, affinity resin, mixed mode chromatography resin,hydrophobic interaction chromatography resin or size exclusionchromatography resin. In one specific exemplary embodiment, thechromatographic resin can be size exclusion chromatography resin.

In some exemplary embodiments, the electrospray ionization massspectrometer can be a nano-electrospray ionization mass spectrometer.

In some exemplary embodiments, the electrospray ionization massspectrometer can be coupled online to a chromatographic system with achromatography resin.

In some exemplary embodiments, the electrospray ionization massspectrometer can be run under native conditions.

In some exemplary embodiments, the chromatographic system can be coupledto the electrospray ionization mass spectrometer using a splitter withat least three paths.

In some exemplary embodiments, the chromatographic system can be coupledto an ultraviolet detector using a splitter with at least three paths.

In some exemplary embodiments, the chromatographic system can be coupledto the electrospray ionization mass spectrometer and an ultravioletdetector using a splitter with at least three paths.

In some exemplary embodiments, the chromatographic system can be coupledto an electrospray ionization mass spectrometer and an ultravioletdetector using a splitter with at least three paths, wherein theelectrospray ionization mass spectrometer is a nano-electrosprayionization mass spectrometer.

In some exemplary embodiments, the chromatographic system can be coupledto an electrospray ionization a mass spectrometer and an ultravioletdetector using a splitter with at least three paths, wherein the massspectrometer can be electrospray ionization mass spectrometer operatingunder native conditions.

In some exemplary embodiments, the chromatographic system can be coupledto an electrospray ionization mass spectrometer and an ultravioletdetector using a splitter with at least three paths, wherein theelectrospray ionization mass spectrometer can be a nano-electrosprayionization mass spectrometer under native conditions.

In some exemplary embodiments, the eluent including the protein orantigen-antibody complex or antibody-drug conjugate from washing theresin can be introduced in an ultraviolet detector through at least onesplitter with at least three paths at a flow rate of about 0.2 mL/min toabout 0.4 mL/min.

In some exemplary embodiments, the mobile phase for washing can have aflow rate of about 0.2 mL/min to about 0.4 mL/min.

In some exemplary embodiments, the mobile phase can comprise a volatilesalt. In some specific embodiments, the mobile phase can compriseammonium acetate, ammonium bicarbonate, or ammonium formate, orcombinations thereof.

In some exemplary embodiments, the mobile phase used can be compatiblewith the mass spectrometer.

In some exemplary embodiments, the sample can comprise about 10 μg toabout 100 μg of the dimer species.

In some exemplary embodiments, the flow rate in the electrosprayionization mass spectrometer can be about 10 nL/min to about 50 nL/min.

In some exemplary embodiments, the electrospray ionization massspectrometer can have a spray voltage of about 0.8 kV to about 1.5 kV.

In some exemplary embodiments, identifying can include proteinsequencing, protein de novo sequencing, identifying post-translationalmodifications, or comparability analysis, or combinations thereof.

In some exemplary embodiments, the sample can also comprise monomer ofproteins.

In some exemplary embodiments, the dimer can comprise a first proteinand a second protein, wherein the first protein and the second proteincan be a therapeutic antibody, an antibody, a monoclonal antibody, apolyclonal antibody, a bispecific antibody, an antibody fragment, afusion protein, or combinations thereof. In one aspect, the antibodyfragment can include Fab fragment, a Fab′ fragment, a F(ab′)2 fragment,a scFv fragment, a Fv fragment, a dsFv diabody, a dAb fragment, a Fd′fragment, a Fd fragment, and an isolated complementarity determiningregion (CDR) region, triabodies, tetrabodies, linear antibodies,single-chain antibody molecules, and multi specific antibodies formedfrom antibody fragments.

In some exemplary embodiments, the dimer species can be aproduct-related impurity present in a co-formulated preparation.

In some exemplary embodiments, the dimer can comprise a first proteinand a second protein, wherein the first protein and the second proteincan have a pI in the range of about 4.5 to about 9.0. In one aspect, theprotein can have a pI of about 4.5, about 5.0, about 5.5, about 5.6,about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 about 6.2, about6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9,about 7.0, about 7.1 about 7.2, about 7.3, about 7.4, about 7.5, about7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 about 8.2,about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about8.9, or about 9.0.

It is understood that the methods are not limited to any of theaforesaid protein, impurity, and column and that the methods foridentifying or quantifying may be conducted by any suitable means.

In some exemplary embodiments, this disclosure provides a systemcomprising a chromatographic column 100 having a chromatography resin,wherein the chromatographic column can be capable of receiving a mobilephase and a sample including a protein, and an electrospray ionizationmass spectrometer 110 (See FIG. 1 ).

In some exemplary embodiments, the chromatographic column 100 can have aresin selected from hydrophobic interaction chromatography resin, anionexchange resin, anion exchange resin, affinity chromatography rein, sizeexclusion chromatography resin, a mixed mode resin, or combinationsthereof.

In some exemplary embodiments, the electrospray ionization massspectrometer 110 can be capable of being coupled to said chromatographiccolumn 100.

In some exemplary embodiments, the electrospray ionization massspectrometer 110 can be capable of being run under native conditions.

In some exemplary embodiments, the electrospray ionization massspectrometer 110 can be a nano electrospray ionization massspectrometer.

In some exemplary embodiments, the electrospray ionization massspectrometer 110 can be a nano electrospray ionization mass spectrometerrun under native conditions.

In some exemplary embodiments, the chromatographic column 100 can becapable of being coupled to the electrospray ionization massspectrometer 100 using a splitter with at least three paths 120.

In some exemplary embodiments, the chromatographic column 100 can becapable of being coupled to an ultraviolet detector 130 using a splitterwith at least three paths 120.

In some exemplary embodiments, the chromatographic column 100 can becapable of being coupled to an ultraviolet detector 130 and theelectrospray ionization mass spectrometer 110 using a splitter with atleast three paths 120.

In some exemplary embodiments, the three way splitter 120 can be capableof being disproportionately split to allow a flow from thechromatographic column 100 to an ultraviolet detector 130 and theelectrospray ionization mass spectrometer 110.

In some exemplary embodiments, the system can be capable of identifyinga dimer species.

An exemplary embodiment of the system in displayed in FIG. 1 . Apost-column splitter with at least three paths is used to enable UV/MSdual detection. The low volume fraction can be directed to the MS whilethe high volume fraction is transferred to the UV detector. Detectionalmost shares the same retention times. Fractions from the UV detectorcan be collected for sample recovery.

It is understood that the system is not limited to any of the aforesaidprotein, chromatography column, mass spectrometer, antibody-drugconjugate, antigen-antibody complex.

This disclosure, at least in part, provides a method for quantifying aheterodimer species in a sample comprising a first protein and a secondprotein, said method comprising immobilizing an antibody specific to thefirst protein on a solid surface, incubating the sample with saidantibody, capturing a precipitated sample, collecting a flow through,treating the precipitated sample with a first compound, treating theflow through with a second compound, mixing the treated precipitatedsample and at least a portion of the treated flow through to form amixture, and analyzing the mixture using a liquid chromatography coupledto a mass spectrometer to quantify the heterodimer species in thesample.

In some exemplary embodiments, the first protein can be an antibody, abispecific antibody, a multispecific antibody, antibody fragment,monoclonal antibody, or an Fc fusion protein monoclonal antibody. Insome exemplary embodiments, the first protein can be a monoclonalantibody.

In some exemplary embodiments, the second protein can an antibody, abispecific antibody, a multispecific antibody, antibody fragment,monoclonal antibody, or a Fc fusion protein. In some exemplaryembodiments, the first protein can be an Fc fusion protein.

In some embodiments, the sample comprising a first protein and a secondprotein can further comprise species selected from homodimer species offirst protein, homodimer species of first protein, heterodimer speciesof first protein and second protein, or combinations thereof.

In some embodiments, the sample comprising a first protein and a secondprotein can further comprise at least one more protein.

In some embodiments, the solid surface can be selected from affinityresins, magnetic beads and coated plates. Non-limiting examples ofaffinity resins include protein A agarose beads, protein G agarosebeads, protein A sepharose beads and protein G sepharose beads.

In some embodiments, the magnetic beads can have a monolayer ofrecombinant streptavidin covalently coupled to the surface.

In some embodiments, the antibody specific to the first protein on asolid surface and the sample can be incubated for several minutes toseveral hours.

In some embodiments, the precipitated sample can be obtained bycentrifugation. In some specific embodiments, the flow through can beobtained by collecting the supernatant.

In some embodiments, the precipitated sample can be re-suspended byheating the re-suspended precipitated sample.

In some embodiments, the precipitated sample can be re-suspended byheating the re-suspended precipitated sample.

In some embodiments, the precipitated sample can undergo elution.

In some embodiments, the precipitated sample and flow through can bereduced by using a reducing agent. In some specific embodiments, thereducing agent can be dithiothreitol.

In some embodiments, the treatment can include alkylation.

In some embodiments, the first compound and the second compound can beisotopes.

In some exemplary embodiments, the at least a portion of the treatedflow through can include about 10% of the treated flow through. In oneaspect, the at least a portion of the treated flow through can ne about1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about8%, about 9%, about 10%, about 15%, about 20%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, about or 100%.

In some embodiments, the first compound and the second compound can belabeled iodoacetamide and unlabeled iodoacetamide respectively. In someother exemplary embodiments, the first compound and the second compoundcan be unlabeled iodoacetamide and labeled iodoacetamide respectively.

In some embodiments, the mixture can be digested before analyzing itusing the liquid chromatography coupled to the mass spectrometer. In oneaspect, the mixture can be digested using a hydrolyzing agent selectedfrom trypsin, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinaseGlu-C, outer membrane protease T (OmpT), immunoglobulin-degrading enzymeof Streptococcus pyogenes (IdeS), chymotrypsin, pepsin, thermolysin,papain, pronase, and protease from Aspergillus saitoi.

In some embodiments, the mixture can be digested using trypsin.

In some embodiments, the mixture can be deglycosylated before analyzingit using the liquid chromatography coupled to the mass spectrometer.

An example of one exemplary embodiment is represented in FIG. 4 . Asshown in FIG. 4 , the mixture can be digested and then split into half.One half can be analyzed using the liquid chromatography coupled to themass spectrometer and the other half can be deglycosylated and analyzedusing the liquid chromatography coupled to the mass spectrometer.

In some embodiments, the liquid chromatography can be reversed-phased(RP), ion exchange (IEX), mixed mode chromatography and normal phasechromatography (NP).

In some exemplary embodiments, the mass spectrometer can be a tandemmass spectrometer.

In some exemplary embodiments, the mass spectrometer can be aelectrospray mass spectrometer.

In some exemplary embodiments, the mass spectrometer can be anano-electrospray mass spectrometer.

In some exemplary embodiments, the method for quantifying a heterodimerspecies in a sample comprising a first protein and a second protein cancomprise immobilizing an antibody specific to the first protein on asolid surface, capturing a precipitated sample and collecting a flowthrough, and treating the captured precipitated sample with a firstcompound and the flow through with a second compound, mixing the treatedprecipitated sample and at least a portion of the treated flow throughto form a mixture, digesting the mixture, and deglycosyalting thedigested mixture.

In some exemplary embodiments, the method for quantifying a heterodimerspecies in a sample comprising a first protein and a second protein cancomprise immobilizing an antibody specific to the first protein on asolid surface, capturing a precipitated sample and collecting a flowthrough, and treating the captured precipitated sample with a firstcompound and the flow through with a second compound, mixing the treatedprecipitated sample and at least a portion of the treated flow throughto form a mixture, digesting the mixture, and analyzing the mixtureusing a liquid chromatography coupled to a mass spectrometer to quantifythe heterodimer species in the sample.

In some exemplary embodiments, the method for quantifying a heterodimerspecies in a sample comprising a first protein and a second protein cancomprise immobilizing an antibody specific to the first protein on asolid surface, capturing a precipitated sample and collecting a flowthrough, and treating the captured precipitated sample with a firstcompound and the flow through with a second compound, mixing the treatedprecipitated sample and at least a portion of the treated flow throughto form a mixture, digesting the mixture, deglycosyalting the digestedmixture, and analyzing the mixture using a liquid chromatography coupledto a mass spectrometer to quantify the heterodimer species in thesample.

In some exemplary embodiments, the method for quantifying a heterodimerspecies in a sample comprising a first protein and a second protein cancomprise analyzing using a liquid chromatography coupled to a massspectrometer to quantify the heterodimer species in the sample, whereinthe mass spectrometer can be a tandem mass spectrometer.

In some exemplary embodiments, the method for quantifying a heterodimerspecies in a sample comprising a first protein and a second protein cancomprise immobilizing an antibody specific to the first protein on asolid surface, capturing a precipitated sample and collecting a flowthrough, and treating the captured precipitated sample with a firstcompound and the flow through with a second compound, and adding areducing agent to the precipitated sample.

In some exemplary embodiments, the method for quantifying a heterodimerspecies in a sample comprising a first protein and a second protein cancomprise immobilizing an antibody specific to the first protein on asolid surface, capturing a precipitated sample and collecting a flowthrough, and treating the captured precipitated sample with a firstcompound and the flow through with a second compound, and adding areducing agent to the flow through.

In some exemplary embodiments, the method for quantifying a heterodimerspecies in a sample comprising a first protein and a second protein cancomprise immobilizing an antibody specific to the first protein on asolid surface, capturing a precipitated sample and collecting a flowthrough, and treating the captured precipitated sample with a firstcompound and the flow through with a second compound, mixing the treatedprecipitated sample and about 10% of the treated flow through to form amixture, and analyzing the mixture using a liquid chromatography coupledto a mass spectrometer to quantify the heterodimer species in thesample.

In some exemplary embodiments, the method for quantifying a heterodimerspecies in a sample comprising a first protein and a second protein cancomprise immobilizing an antibody specific to the first protein on asolid surface, capturing a precipitated sample and collecting a flowthrough, and treating the captured precipitated sample with a firstcompound and the flow through with a second compound, mixing the treatedprecipitated sample and about 10% of the treated flow through to form amixture, digesting the mixture, and analyzing the mixture using a liquidchromatography coupled to a mass spectrometer to quantify theheterodimer species in the sample.

In some exemplary embodiment, the method for quantifying a heterodimerspecies in a sample can comprise a first protein and a second protein,wherein the sample can be stressed by subjecting it a condition selectedfrom a group consisting of cool-white light exposure, hydrogen peroxideexposure, ultraviolet light exposure, heat, or combinations thereof,including forced degradation under ICH guidelines.

In some embodiments, the % second protein heterodimer in a samplecomprising a first protein and a second protein, wherein the firstprotein is treated with an unlabeled compound and the second protein istreated with a labeled compound, can be calculated using the formulastated below:

${{{second}{protein}{heterodimer}\%} = {\frac{{amount}{of}{second}{protein}{as}{heterodimer}}{{total}{amount}{of}{second}{protein}} = \frac{{area}{light}}{{{area}{light}} + {{area}{heavy}}}}},$

wherein area light is the area under the curve of MS peaks determine forthe second protein treated with an unlabeled compound and area heavy isthe area under the curve of MS peaks determine for the second proteintreated with an labeled compound. The method can also be implementedsuch that the first protein is treated with a labeled compound and thesecond protein is treated with a unlabeled compound.

In some embodiments, the % second protein heterodimer in a samplecomprising a first protein and a second protein, wherein the firstprotein is treated with an unlabeled compound and the second protein istreated with a labeled compound, can be calculated using the formulastated below:

${{{second}{protein}{heterodimer}\%} = {\frac{{amount}{of}{second}{protein}{as}{heterodimer}}{{total}{amount}{of}{second}{protein}} = \frac{{area}{light}}{{{area}{light}} + {10\left( {{area}{heavy}} \right)}}}},$

wherein area light is the area under the curve of MS peaks determine forthe second protein treated with an unlabeled compound and area heavy isthe area under the curve of MS peaks determine for the second proteintreated with an labeled compound. The method can also be implementedsuch that the first protein is treated with a labeled compound and thesecond protein is treated with a unlabeled compound.

The consecutive labeling of method steps as provided herein with numbersand/or letters is not meant to limit the method or any embodimentsthereof to the particular indicated order.

Various publications, including patents, patent applications, publishedpatent applications, accession numbers, technical articles and scholarlyarticles are cited throughout the specification. Each of these citedreferences is herein incorporated by reference, in its entirety and forall purposes.

The disclosure will be more fully understood by reference to thefollowing Examples, which are provided to describe the disclosure ingreater detail. They are intended to illustrate examples and should notbe construed as limiting the scope of the disclosure.

EXAMPLES

Materials and reagents. Water was purchased from Honeywell (Muskegon,Mich.). Ammonium acetate was purchased from Sigma-Aldrich (St Louis,Mo.). 1 M Tris-HCl, pH 7.5 was purchased from Teknova (Hollister,Calif.). Fused silica tubing (inner Diameter (ID) 150 μm, outer diameter(OD) 360 μm), 3-way connector and sleeve were purchased from IDEX (OakHarbor, Wash.). PicoTip EMITTER SilicaTip (FS360-20-10-D-20-7CT) waspurchased from New Objective (Woburn, Mass.). ACQUITY UPLC Protein BEHSEC Column, 200 Å, 1.7 μm, 4.6×300 mm was purchased from Waters(Milford, Mass.). Hot pocket column heater was purchased fromThermo-Fisher (Waltham, Mass.). All reagents were used withoutadditional purification.

Online SEC-nano-ESI-MS analysis. ACQUITY UPLC I class system (Waters,Milford, Mass.) was coupled to Q Exactive HF hybrid quadrupole-Orbitrapmass spectrometer (Thermo Scientific, Bremen, Germany) for all onlineSEC-nano-ESI-MS analyses. ACQUITY UPLC Protein BEH SEC Column (200 Å,1.7 μm, 4.6×300 mm) was set at 30° C. and used for mAbs and ADCsseparation. Mobile phase was 100 mM ammonium acetate at pH 6.8. Eachseparation was 30 minutes with a flow rate of 0.3 mL/min, and theinjection amount was set to 40 μg. A three-way splitter (T-splitter) wasconnected after the SEC column. Fused silica tubing (L: 140 cm, ID: 150μm) and SilicaTip (L: 5 cm, ID: 10 μm) were connected to the T-splitter.The high volume fraction was transferred to the UV detector via fusedsilica tubing, while the low volume fraction was diverted to the MS viaa SilicaTip. The following MS parameters were used for onlineSEC-nano-ESI-MS data acquisition. Each acquisition was 25 minutesbeginning immediately after sample injection. Samples were ionized inpositive mode with 3 kV spray voltage, 200° C. capillary temperature,and 70 S-lens RF level. In-source CID was set at 75 eV. Full MS scanswere acquired at 15 K resolving power with mass range between m/z2000-8000. A maximum injection time of 100 ms, automatic gain controltarget value of 3e6, and 10 microscans were used for full MS scans.

Data analysis. Protein Metrics Intact Mass software was used for rawdata deconvolution. Thermo Xcalibur Qual Browser was used for extractedion chromatogram analysis. Microsoft Excel was used for DAR calculationof ADCs.

Example 1

1.1 Online SEC-Nano-ESI-MS Instrumentation

SEC and MS technologies are routinely used for characterizing proteinsamples. SEC allows for the isolation and characterization of proteinsunder conditions that minimize changes in protein structure, while MSpermits the identification of individual components in complex samples.Combining the individual capabilities of SEC and MS into a singleplatform would be highly desirable, but has proven challenging becausethe high flow rate and nonvolatile salts used for SEC analyses areincompatible with native MS. To overcome this limitation, reduction ofsolvent and salt intake into the MS by splitting the flow of eluate fromthe SEC using a post-column T-splitter was performed (See FIG. 1 ). TheT-splitter was then connected to the MS via a SilicaTip and, inparallel, to a UV detector via fused silica tubing. This arrangementenabled simultaneous, dual UV/MS detection of SEC elutes. By varying thelength and diameter of the fused silica tubing, flow rate to the MS viathe SilicaTip could be regulated (e.g. longer/narrower tubing cangenerate higher resistance causing increased flow to the SilicaTip andMS). Protein samples were separated with a 4.6 mm SEC column using a 0.3mL/min flow rate. The fused silica tubing connecting T-splitter and UVdetector with a length of 140 cm and an inner diameter of 150 μmresulted in a desirable flow rate of ˜1 μL/min to the SilicaTip. Thelength and diameter of the fused silica tubing also enabled nearsynchronous detection of molecules by the UV and MS.

1.2 Co-Formulated Preparation

A formulation comprising mAb1, mAb2, and mAb3 was used for theexperiment. Six dimer species can be possibly present in the formulation(See FIG. 5 ). Samples were stressed under various conditions, includingconditions provided in ICH guidelines such as photo stability testing(UV 1xICH and CW 1xICH).

1.3 Results

Five dimers (mAb1-mAb1, mAb1-mAb2, mAb2-mAb2, mAb2-mAb3, and mAb3-mAb3)were successfully differentiated using the system illustrated in 1.1using both the extracted ion chromatograms (XIC) and the molecularweight measurement as shown in FIGS. 6 and 7 . The only heterodimer notidentified was the mAb1-mAb3 since its molecular weight was very closeto homodimer mAb2-mAb2 (different by 15 Da) (See FIG. 7 ).

Example 2

2.1 Online SEC-Nano-ESI-MS Instrumentation

The Instrumentation as Illustrated in 1.1 was Used.

2.2 Co-Formulated Preparation

A formulation comprising mAb1 and a fusion protein was used for theexperiment. Three dimer species can be possibly present in theformulation (See FIG. 8 ). The presence of N-glycans in the fusionprotein significantly complicates the intact mass analysis.

A co-formulation comprising mAb1 and fusion protein in the ratio of120:40, a co-formulation comprising mAb1 and fusion protein in the ratioof 60:40, a formulation of mAb1 (120 mg/mL), a formulation of mAb1 (60mg/mL), and a formulation of fusion protein (40 mg/mL), was subjected tofour stress conditions: (a) cool-white light (CW), (b) hydrogen peroxidefor 18 hours, (c) ultraviolet light (UV), and (d) heating at 37° C. for28 hours. The stress conditions, included conditions provided by ICHguidelines.

The formulations and co-formulations subjected to the heating at 37° C.for 28 hours and treatment with hydrogen peroxide for 18 hours led to amild increase in high molecular weight species (dimers or otheraggregates). On the contrary, the formulations and co-formulationssubjected treatment with cool-white light and ultraviolet lightcomprised high levels of high molecular weight species.

2.3 Identification of Dimer Species

The co-formulation comprising mAb1 and fusion protein in the ratio of120:40 subjected to cool-white light was analyzed using the systemillustrated in 1.1. FIG. 9 shows the extracted ion chromatogram (XIC) ofthe co-formulation analyzed using SEC-nano-ESI-MS. The method leads todifferentiation of the monomer species and dimer species.

The application of charge reduction technique greatly improved the massmeasurement of a protein ingredient that is highly heterogeneous inmolecular weight as exemplified in FIG. 10 .

An online SEC-nano-electrospray ionization (nano-ESI)-MS platform withdual ultraviolet (UV) and MS detection was developed. The utility ofthis platform was validated by identifying homodimer and heterodimerspecies in a co-formulated preparation.

The three way splitter was used to disproportionately split SEC eluatesto a MS and UV detector, with the low-volume fraction directed to the MSand the high-volume fraction directed to the UV detector. The currentplatform enabled complementary dual detection by UV and native-MS, withthe possibility of fraction collection, and can be applied to thecharacterization of dimer species. Further modifications to this onlineSEC-nano-ESI-MS platform, such as changing the column chemistry or usinga Q Exactive UHMR instrument, would allow it to be adapted for otherapplications such as analyzing charge variants or very large proteincomplexes. The method described herein unlocked the possibility ofcombining high salt separation techniques (i.e. HIC, WCX) with massspectrometry-based detection. In conclusion, the online SEC-nano-ESI-MSplatform could be broadly applied to the analysis of proteinbiopharmaceuticals for a variety of applications.

Example 3

3.1 Sample Preparation

A co-formulation comprising mAb1 and fusion protein 1 was used. Thedifferent samples (50 μL) used for the experiment are illustrated inTable 1. The negative control (sample 3) was prepared by mixing thestressed fusion protein 1 (sample 1) and stressed mAb1 (sample 1) rightbefore analysis, wherein the samples were stressed using ultravioletlight as provided under ICH guidelines. The other negative control(sample 7) was prepared by mixing the stressed fusion protein 1 (sample6) and stressed mAb1 (sample 5) right before analysis, wherein thesamples were stressed using cool-white light as provided under ICHguidelines.

TABLE 1 % fusion % Samples (stress % mAb1 protein 1 Total % %conditions) Conc. Info Monomer Monomer Monomer HMW LMW 1 mAb1 (UV) mAb1(120 mg/mL) 80.25 NA 80.25 18.51 1.24 2 fusion protein 1 fusion protein1 (40 NA 81.03 81.03 18.97 0 (UV) mg/mL) 3 Negative Mixture of the aboveControl (UV) two 4 mAb1 + fusion 120:40, mAb1:fusion 67.64 14.75 82.3916.63 0.98 protein 1 (UV) protein 1 5 mAb1 (CW) mAb1 (120 mg/mL) 73.75NA 73.75 24.91 1.33 6 fusion protein 1 fusion protein 1 (40 NA 81.7881.78 18.23 0 (CW) mg/mL) 7 Negative Mixture of the above Control (CW)two 8 mAb1 + fusion 120:40, mAb1:fusion 62.01 12.87 74.88 24.15 0.99protein 1 (CW) protein 1

3.2 Immunoprecipitation

The immunoprecipitation was carried out as shown in FIG. 11 . In sixcentrifugation tubes, 0.5 ml Dynabeads MyOne Streptavidin T1 (magneticbeads) and 100 μg Anti-mAb1 antibody was added. The samples 1, 2, 4, 5,6, and 8 were added to individual tubes and incubated for 30 minutes atroom temperature by gently mixing on a suitable shaker. The tubes wereplaced on a magnetic stand for 3 minutes to ensure that all of the beadsare collected. The supernatant was removed and 0.4 mL of 100 mM ofAmmonium acetate buffer was added. The tubes were incubated for 5minutes on a Thermomixer at 800 rpm. This step was repeated twice (total3 times). All the supernatant, 1.2 mL of supernatant, was collected in a1.5 mL tube (and marked as FT1-flow through). The magnetic beads werewashed with 0.4 mL 10% Acetonitrile using a Thermomixer at 800 rpm. Thebeads were placed on the magnetic stand for 3 minutes to ensure that allof the beads are collected. The supernatant was removed between washes.This step was repeated three times (total 4 times). All of thesupernatant was collected—1.2 mL of supernatant. In a 1.5 mL tube, markas FT2; A 0.2 mL of elution buffer (50% ACN, 0.1% formic acid) was addedand incubated at room temperature for 5 min on a Thermomixer at 1000rpm. All the supernatant was collected into a tube and marked as PD-pulldown.

3.3 Analysis

The analysis of the sample involved the following steps: Add 20 uL of 8M urea in 100 mM Tris-HCl (pH 7.5) to each sample tube. Add 1 μL of 100mM dithiothreitol (DTT) in Milli-Q water to each sample, vortex to mixwell and incubate at 50° C. for 30 minutes. After incubation, add 1.1 uLof 200 mM light labeled iodoacetamide (light-IAA) in Milli-Q water tothe pull down, and add 1.1 uL of 200 mM heavy labeled iodoacetamide(heavy-IAA) in Milli-Q water to the flow through. Incubate both pulldown and flow through at room temperature in the dark for 30 minutes.Then, the entire pull down and about 10% of the flow through were mixedand digested by adding 4 μg of Trypsin and incubated for overnight at37° C. The digested mixture was analyzed using LC-MS (Waters ACQUITYUPLC system and Thermo Q Exactive mass spectrometer).

FIG. 12 shows the mass to charge ratios of the pull-down andflow-through obtained for samples 7 and 8. The pull-down fractioncontains fusion protein 1 in heterodimer and flow-through fractioncontains fusion protein 1 present in monomer and homodimer. FIG. 13shows the calculated percentage of fusion protein 1 heterodimer. Thecalculation was carried out using the formula shown in FIG. 4 . For someof the peptides used to quantify the fusion protein 1, the mass tocharge ratio charts in samples 3,4, 7 and 8 are shown in FIGS. 14-17 .The peptide 1, peptide 2, peptide 3, and peptide 4 in the figuresrepresent peptide²⁵ELVIPCR³¹, peptide ⁷³EIGLLTCEATVNGHLYK⁸⁹,peptide¹²⁰LVLNCTAR¹²⁷, and peptide ¹⁷⁸SDQGLYTCAASSGLMTK¹⁹⁴ respectively.

The % fusion protein 1 heterodimer was converted into heterodimer % byfollowing the method as shown in FIG. 18 . Based on the calculations forheterodimer % represented in FIG. 18 , the heterodimer % in the sample 4and sample 8 was found to be 6.4% and 8.5% respectively. The low levelsof “heterodimer signal” from negative control samples (samples 3 and 7)could be attributed from interaction between mAb1 and fusion protein 1after mixing and non-specific interaction between mAb1 and the affinityresin. The heterodimer % was consistent with the overall HMW %.

Example 4

To evaluate the quantitation limit and linearity of the fusion proteinheterodimer by immunoprecipitation, samples with sequentially dilutedcool-white stressed co-formulation preparation were used.

The testing samples prepared as listed in Table 2. A co-formulationcomprising mAb1 and fusion protein 1 was used and stressed using acool-white light. The negative control mixed sample was prepared bymixing the stressed fusion protein 1 (sample 6) and stressed mAb 1(sample 5) right before analysis. The different testing samples used forthe experiment were prepared as illustrated in Table 2.

TABLE 2 Mix solutions Negative Co-formulated- control CW sample Mixedsample Final Tested Sample (μL) (μL) Volume 1 Co-formulated-CW-1 20 0 202 Co-formulated-CW-½ 10 10 20 3 Co-formulated-CW-¼ 5 15 20 4Co-formulated-CW-⅛ 2.5 17.5 20 5 Co-formul ated-CW- 1/16 1.25 18.75 20 6Negative control 0 20 20

The immunoprecipitation and analysis was carried out as illustrated in3.2 and 3.3. The chart of fusion protein heterodimer % in the testedsamples normalized to fusion protein heterodimer % in the tested samplewith no dilution is shown in FIG. 19 . Further, the plot of fusionprotein heterodimer % vs. % cool-white stressed sample in the mixedsample is shown in FIG. 20 , for three of peptides used to quantifyfusion protein 1, wherein the sample at dilution 1 represents the neatcool white-stressed co-formulated sample. The quantitation methodmaintained a good linearity at least as low as 0.8% of heterodimer (thelowest value tested).

Example 5

To evaluate the quantitation of heterodimer in ambient conditionsstressed co-formulated samples, samples from a co-formulated preparationcomprising mAb1 and fusion protein 1 were stored at 25° C. for 12 monthsand 5° C. for 46 months. The immunoprecipitation and analysis wascarried out as illustrated in 3.2 and 3.3. The quantitation calculatedin the samples is shown in Table 3 and FIG. 21 , wherein the differentfusion protein 1 peptides were used for quantitation.

TABLE 3 Mix-CW (Negative Quantitation 25° C. 12 m 5° C. 46 m Control)ELVIPCR 5.10% 3.38% 0.40% SDQGLYTCAASSGLMTK 5.18% 3.56% 0.36%EIGLLTCEATVNGHLYK 4.89% 3.47% 0.44% Average_(heterodimer) % 5.06% 3.47%0.40% % heterodimer (Calculated) 2.78% 1.91% 0.22% In UV Peak Areas %HMW 6.40% 5.26%

Thus, a new immunoprecipitation method was developed for thequantitation of heterodimer level in samples obtained from co-formulatedpreparations. It was successfully applied to quantitate the level ofheterodimer in both UV/CW stressed and ambient condition stressedsamples from co-formulated preparations.

What is claimed is:
 1. A method for identifying at least one dimerspecies, said method comprising: contacting a sample including the dimerspecies to a chromatographic system having a size-exclusionchromatography resin; washing said size-exclusion chromatography resinusing a mobile phase to provide an eluent including the dimer species;and identifying the dimer species in said eluent using an electrosprayionization mass spectrometer under native conditions.
 2. The method ofclaim 1, wherein the electrospray ionization mass spectrometer iscoupled online to the chromatographic system having the size-exclusionchromatography resin.
 3. The method of claim 1, wherein the electrosprayionization mass spectrometer is a nano-electrospray ionization massspectrometer.
 4. The method of claim 1, wherein at least one splitterwith at least three paths is used to allow fluid communication with theelectrospray ionization mass spectrometer and the chromatographic systemhaving the size-exclusion chromatography resin.
 5. The method of claim1, wherein at least one splitter with at least three paths is used toallow fluid communication between an ultraviolet detector to thechromatographic system having the size-exclusion chromatography resin.6. The method of claim 5, wherein the eluent from washing thesize-exclusion chromatography resin is introduced in the ultravioletdetector through the at least one splitter at a flow rate of about 0.2mL/min to about 0.4 mL/min.
 7. The method of claim 1, wherein the mobilephase used to wash the size-exclusion chromatography resin comprisesammonium acetate.
 8. The method of claim 1, wherein the mobile phaseused to wash the size-exclusion chromatography resin comprises avolatile salt.
 9. The method of claim 1, wherein the mobile phase usedto wash the size-exclusion chromatography resin has a flow rate of about0.2 mL/min to about 0.4 mL/min.
 10. The method of claim 1, wherein anamount of the sample including the dimer species contacted to thechromatography system is about 10 μg to about 100 μg.
 11. The method ofclaim 1, wherein the eluent provided from washing the size-exclusionchromatography resin is introduced in the electrospray ionization massspectrometer, wherein a flow rate of electrospray from the electrosprayionization is about 10 nL/min to about 50 nL/min.
 12. The method ofclaim 1, wherein the eluent provided from washing the size-exclusionchromatography resin is introduced in the electrospray ionization massspectrometer, wherein a spray voltage of electrospray is about 0.8 kV toabout 1.5 kV.
 13. The method of claim 1, wherein the sample comprises atleast two dimer species.
 14. The method of claim 1, wherein the samplecomprises a homodimer species.
 15. The method of claim 1, wherein thesample comprises a heterodimer species.
 16. The method of claim 1,wherein the sample is obtained from a co-formulated preparation.
 17. Amethod for quantifying a heterodimer species, said method comprising:immunoprecipitating the heterodimer species; and quantifying theheterodimer species by using a stable isotope labeling method followedby a liquid chromatography coupled to a mass spectrometer.
 18. A methodfor quantifying a heterodimer species in a sample comprising a firstprotein and a fusion protein, said method comprising: immobilizing anantibody specific to the first protein on a solid surface; incubatingthe sample with said antibody; capturing a precipitated sample;collecting a flow through; treating the precipitated sample with a firstcompound; treating the flow through with a second compound; mixing thetreated precipitated sample and at least a portion of the treated flowthrough to form a mixture; and analyzing the mixture using a liquidchromatography coupled to a mass spectrometer to quantify theheterodimer species in the sample.
 19. The method of claim 18, whereinthe first compound is an isotope of the second compound.
 20. The methodof claim 19, wherein the first compound is iodoacetamide.
 21. The methodof claim 18, wherein the sample is stressed by subjecting it to acondition selected form a group consisting of cool-white light exposure,hydrogen peroxide exposure, ultraviolet light exposure, heat, orcombinations thereof.